1. Field of the Invention
The present invention concerns methods and means for the identification and use of modulators of xcex2-amyloid (Axcex2) levels obtained by the proteolytic processing of the xcex2-amyloid precursor protein, APP.
2. Description of the Related Art
A number of important neurological diseases, including Alzheimer""s disease (AD), cerebral amyloid angiopathy (CAA), and prion-mediated diseases are characterized by the deposition of aggregated proteins, referred to amyloid, in the central nervous system (CNS) (for reviews, see Glenner et al., J. Neurol. Sci. 94:1-28 [1989]; Haan et al., Clin. Neurol. Neurosurg. 92(4):305-310 [1990]). These highly insoluble aggregates are composed of nonbranching, fibrillar proteins with the common characteristic of xcex2-pleated sheet conformation. In the CNS, amyloid can be present in cerebral and meningeal blood vessels (cerebrovascular deposits) and in the brain parenchyma (plaques). Neuropathological studies in human and animal models indicate that cells proximal to amyloid deposits are disturbed in their normal functions (Mandybur, Acta Neuropathol. 78:329-331 [1989]; Kawai et al., Brain Res. 623:142-146 [1993]; Martin et al., Am. J. Pathol. 145:1348-1381 [1994]; Kalaria et al., Neuroreport 6:477-480 [1995]; Masliah et al., J. Neurosci. 16:5795-5811 [1996]; Selkoe, J. Biol. Chem. 271:18295-18298 [1996]; Hardy, Trends Neurosci 20:154-159 [1997]).
AD and CAA share biochemical and neuropathological markers, but differ somewhat in the extent and location of amyloid deposits as well as in the symptoms exhibited by affected individuals. The neurodegenerative process of AD, the most common neurodegenerative disorder worldwide, is characterized by the progressive and irreversible deafferentation of the limbic system, association neocortex, and basal forebrain accompanied by neuritic plaque and tangle formation (for a review, see Terry et al., xe2x80x9cStructural alteration in Alzheimer""s disease,xe2x80x9d In: Alzheimer""s disease, Terry et al. Eds., 1994, pp. 179-196, Raven Press, New York). Dystrophic neurites, as well as reactive astocytes and microglia, are associated with these amyloid-associated neuritic plaques. Although the neuritic population in any given plaque is mixed, the plaques generally are composed of spherical neurites that contain synaptic proteins, APP (type I), and fusiform neurites containing cytoskeletal proteins and paired helical filaments (PHF; type II).
CAA patients display various vascular syndromes, of which the most documented is cerebral parenchymal hemorrhage. Cerebral parenchymal hemorrhage is the result of extensive amyloid deposition within cerebral vessels (Hardy, Trends Neurosci 20:154-159 [1997]; Haan et al., Clin. Neurol. Neurosurg. 92:305-310 [1990]; Terry et al., [1994] supra; Vinters, Stroke 18:211-224 [1987]; Itoh et al., J. Neurosurgical Sci. 116:135-141 [1993]; Yamada et al., J. Neurol. Neruosurg. Psychiatry 56:543-547 [1993]; Greenberg et al., Neurology 43:2073-2079 [1993]; Levy et al., Science 248:1124-1126 [1990]). In some familial CAA cases, dementia was noted before the onset of hemorrhages, suggesting the possibility that cerebrovascular amyloid deposits may also interfere with cognitive functions.
Both AD and CAA are characterized by the accumulation of senile plaques in the brains of the affected individuals. The main amyloid components is the amyloid xcex2 protein (Axcex2), also referred to as amyloid xcex2 or xcex2-amyloid peptide, derived from proteolytic processing of the xcex2-amyloid precursor protein, xcex2-APP or simply APP. For review in connection with AD see, Selkoe, D. J. Nature 399: A23-A31 (1999). Axcex2 is produced by proteolytic cleavage of an integral membrane protein, termed the xcex2-amyloid precursor protein (xcex2APP).
The Axcex2 peptide, which is generated from APP by two putative secretases, is present at low levels in the normal CNS and blood. Two major variants, Axcex21-40 and Axcex21-42 are produced by alternative carboxy-terminal truncation of APP (Selkoe et al. [1988] Proc. Natl. Acad. Sci. USA 85:7341-7345; Selkoe [1993] Trends Neurosci 16:403-409). Axcex21-42 is the more fibrillogenic and more abundant of the two peptides in amyloid deposits of both AD and CAA. In addition to the amyloid deposits in AD cases described above, most AD cases are also associated with amyloid deposition in the vascular walls (Hardy [1997], supra; Haan et al. [1990], supra; Terry et al., [1994] supra; Vinters [1987], supra; Itoh, et al. [1993], supra; Yamada et al. [1993], supra; Greenberg et al. [1993], supra; Levy et al. [1990], supra). These vascular lesions are the hallmark of CAA, which can exist in the absence of AD.
The precise mechanisms by which neuritic plaques are formed and the relationship of plaque formation to the AD-associated, and CAA-associated neurodegenerative processes are not well defined. However, evidence indicates that dysregulated expression and/or processing of APP gene products or derivatives of these gene products are involved in the pathophysiological process leading to neurodegeneration and plaque formation. For example, missense mutations in APP are tightly linked to autosomal dominant forms of AD (Hardy [1994] Clin. Geriatr. Med. 10:239-247; Mann et al. [1992] Neurodegeneration 1:201-215). The role of APP in neurodegenerative diseases is further implicated by the observation that persons with Down""s syndrome who carry an additional copy of the human APP (HAPP) gene on their third chromosome 21 show an overexpression of hAPP (Goodison et al. [1993] J. Neuropathol. Exp. Neurol. 52:192-198; Oyama, et al. [1994] J. Neurochem, 62:1062-1066) as well as a prominent tendency to develop AD-type pathology early in life (Wisniewski et al. [1985] Ann. Neurol. 17:278-282). Mutations in Axcex2 are linked to CAA associated with hereditary cerebral hemorrhage with amyloidosis (Dutch HCHWA) (Levy, et al. [1990], supra), in which amyloid deposits preferentially occur in the cerebrovascular wall with some occurrence of diffuse plaques (Maat-Schieman et al. [1994] Acta Neuropathol 88:371-8; Wartendorff et al. [1995] J. Neurol. Neurosurg. Psychiatry 58:699-705). A number of HAPP point mutations that are tightly associated with the development of familial AD encode amino acid changes close to either side of the Axcex2 peptide (for a review, see, e.g., Lannfelt et al. [1994] Biochem. Soc. Trans. 22:176-179; Clark et al. [1993] Arch. Neurol. 50:1164-1172). Finally, in vitro studies indicate that aggregated Axcex2 can induce neurodegeneration (see, e.g., Pike et al. (1995) J. Neurochem. 64:253-265).
APP is a glycosylated, single-membrane-spanning protein expressed in a wide variety of cells in many mammalian tissues. Examples of specific isotypes of APP which are currently known to exist in humans are the 695-amino acid polypeptide (APP695) described by Kang et al. (1987) Nature 325:733-736, which is designated as the xe2x80x9cnormalxe2x80x9d APP. A 751-amino acid polypeptide (APP751) has been described by Ponte et al. (1988) Nature 331:525-527 and Tanzi et al. (1988) Nature 331:528-530. A 770-amino acid isotype of APP (APP770) is described in Kitaguchi et al. (1988) Nature 331:530-532. A number of specific variants of APP have also been described having mutations which can differ in both position and phenotype. A general review of such mutations is pivoted in Hardy (1992) Nature Genet. 1:233-235. A mutation of particular interest is designated the xe2x80x9cSwedishxe2x80x9d mutation where the normal Lys-Met residues at positions 595 and 596 are replaced by Asn-Leu. This mutation is located directly upstream of the normal P-secretase cleavage site of APP, which occurs between residues 596 and 597 of the 695 isotype.
APP is post-translationally processed by several proteolytic pathways resulting in the secretion of various fragments or intracellular fragmentation and degradation. F. Checler, J. Neurochem. 65:1431-1444 (1995). The combined activity of xcex2-secretase and xcex3-secretase on APP releases an intact Axcex2-amyloid peptide (Axcex2), which is a major constituent of amyloid plaques. Initial cleavage of APP by xcex2-secretase generates soluble APPxcex2 and membrane-associated xcex2-CTF that can be further processed by xcex3-secretase to generate a 40 or a 42 amino acid peptide (Axcex240 or Axcex242). Alternatively, APP processing by xcex1-secretase leads to the formation of soluble APPxcex1 and membrane associated xcex1-CTF the latter being a substrate for xcex3-secretase to generate the non-amyloidogenic p3. Axcex2 is an approximately 43 amino acid peptide, which comprises residues 597-640 of the 695 amino acid isotype of APP. Internal cleavage of Axcex2 by a xcex1-secretase inhibits the release of the full-length Axcex2 peptide. Although the extent of pathogenic involvement of the secretases in AD progression is not fully elucidated, these proteolytic events are known to either promote or inhibit Axcex2 formation, and thus are thought to be good therapeutic candidates for AD.
The polytopic transmembrane protein presenilin has been strongly implicated in xcex3-secretase activity (for review see Haass and De Strooper, Science 286: 916-919 [1999]). Mutagenesis of two transmembrane aspartates of presenilin led to the inactivation of xcex3-secretase activity in cellular assays (Wolfe et al., Nature 398: 513-517 [1999]). As a result, both xcex1- and xcex2-CTFs accumulated and Axcex2 formation was significantly decreased. Similar effects were seen upon inhibition of xcex3-secretase using substrate analogs (Wolfe et al., J. Med. Chem. 41: 6-9 [1998]). While it remains to be determined whether presenilin is sufficient as xcex3-secretase or whether it requires another unique co-factor of so far unknown nature to exert its function Presenilin 1 and xcex3-secretase activity have recently been shown to co-precipitate from membrane extracts (Li et al. Proc. Natl. Acad Sci. USA 97(11):6138-43 [2000]).
As discussed above, there are at least two proteases involved in the generation of Axcex2, referred to as xcex2- and xcex3-secretases (Citron et al., Neuron 17:171-179 [1996]; Seubert et al., Nature 361:260-263 [1993]; Cai et al., Science 259:514-516 [1993]; and Citron et al., Neuron 14:661-670 [1995]). There have been intense efforts in recent years to identify and characterize these enzymes. Recently five independent groups have reported cloning and characterization of genes corresponding to a xcex2-secretase (Vassar et al., Science 286: 735-741 [1999]; Yan et al., Nature 402: 533-537 [1999]; Sinha et al., Nature 402: 537-540 [1999]; Hussain et al., Mol. Cell. Neurosci. 14: 419-427 [1999]; Lin et al. Proc. Natl. Acad. Sci. USA 97: 1456-1460 [2000]). The membrane-bound aspartyl protease has been variously referred to as xcex2-site APP-cleaving enzyme (BACE), Aspartyl protease-2 (Asp2), memapsin 2 or simply as xcex2-secretase . However, the deduced amino acid sequence of the polypeptide chain reported by all five groups is identical. The cloned enzyme possesses many of the characteristics expected of an authentic xcex2-secretase. In particular, BACE overexpression resulted in an increase in both xcex2-NTF and Axcex2 levels while suppression of BACE with antisense oligonucleotides led to a significant reduction of these cleavage products. As predicted for the genuine xcex2-secretase, the Swedish double mutant of APP (APPsw, Mullan et al., Nature Genetics 1: 345-347 [1992]; Citron et al, Nature 360: 672-674 [1992]; Cai et al., Science 259: 514-516 [1993]) was cleaved more efficiently by BACE. Taken together, these results have led to the notion that BACE is the main xcex2-secretase activity.
A close homolog of BACE, designated DRAP or BACE2, has been described recently (Acquati et al., FEBS Lett. 468: 59-64 [2000], GenBank accession numbers for the human and mouse cDNA sequences: AF050171 and AF051150, respectively; Bennett et al., J. Biol. Chem. 275:37712-7 [2000]). BACE and BACE2 share 64% amino acid similarity but the role of BACE2 in APP processing has not yet been elucidated. Strikingly, BACE2 expression in brain appears to be very low and this observation has contributed to the assumption that BACE2""s role in xcex2-secretase cleavage might only be minor (Bennett et al., ibid).
Experimental data disclosed herein confirm that BACE2 indeed possesses xcex2-secretase activity when reconstituting xcex2-secretase cleavage in a cell-free assay using wild-type (wt) or Swedish mutant forms of APP751 as a substrate. However, this activity is weaker than the xcex2-secretase activity of BACE. The invention is further based on the unexpected finding that while BACE2 overexpression in HEK293 cells had a moderate effect on xcex2-NTF formation, it strikingly suppressed Axcex2 production in either the presence or absence of additional exogenous copies of BACE. BACE2 also modulated Axcex2 levels in neuronal SKN cells and thus its effect was not restricted to non-neuronal HEK293 cells. The suppression of Axcex2 production by BACE2 did not appear to require its ability to cleave at the xcex2-secretase site. Axcex2 levels were similarly suppressed in cells carrying a C-terminal 100 amino acids fragment of amyloid precursor protein (APP) truncated to mimic xcex2-secretase cleavage. It is suggested that BACE2 functions as a modulator of Axcex2 production by promoting the alternative non-amyloidogenic APP processing pathway such as that mediated by xcex1-secretase activity.
In one aspect, the invention concerns a method of modulating the enzymatic production of Axcex2-amyloid peptide (Axcex2) from xcex2-amyloid precursor protein (APP) or a fragment thereof by contacting said APP or APP fragment with a BACE2 polypeptide or an agonist or antagonist thereof. In a specific embodiment, the method concerns the inhibition of Axcex2 production from APP or an APP fragment by using BACE2 or an agonist of BACE2.
In another aspect, the invention concerns a method of inhibiting the formation of an Axcex2-amyloid peptide (Axcex2) from Axcex2-amyloid precursor protein (APP) or a fragment thereof by contacting APP of an APP fragment with a BACE2 polypeptide or an agonist thereof.
In yet another aspect, the invention concerns a method of inhibiting the release of a full-length xcex2-amyloid (Axcex2) polypeptide from xcex2-amyloid precursor protein (APP) or a fragment thereof, comprising cleaving said APP or APP fragment by a BACE2 polypeptide or an agonist thereof at a site interfering with ""-secretase processing of the APP or APP fragment.
In all aspects, APP may, for example, be a native sequence human APP including the 695-amino acid isotype and the isotype containing the so called Swedish mutation. The APP fragment specifically includes, without limitation, xcex2-CTF. The methods may be performed in the presence of an xcex1-secretase activity and/or a xcex3-secretase activity and/or a xcex2-secretase activity other than BACE2. The additional xcex2-secretase activity may, for example, be due to the presence of an enzyme having a pH optimum at about pH 6.5-7.0, and an estimated molecular weight of about 32-39 kDa as calculated from radiation inactivation analysis of HEK293 cell membrane extracts, or about 20-26 kDa as calculated from radiation inactivation analysis of human brain samples, with a candidate compound. Alternatively or in addition, the additional xcex2-secretase activity may be due to the presence of a xcex2-secretase enzyme having a pH optimum at about pH 4.5-5.0 and an estimated molecular weight of about 50-60 kDa as calculated from radiation inactivation analysis of HEK293 cell membrane extracts or human brain samples (BACE1). These xcex2-secretase activities are further disclosed and characterized in co-pending application Ser. No. 09/566,746 filed on May 9, 2000 for Novel xcex2-Secretase and Modulation of xcex2-Secretase Activity the entire disclosure of which is hereby expressly incorporated by reference.
In a further aspect, the invention concerns a method for identifying a modulator of the enzymatic production of xcex2-amyloid peptide (Axcex2) from xcex2-amyloid precursor protein (APP) or a fragment thereof, comprising contacting APP or an APP fragment and BACE2 with a candidate compound and monitoring the effect of the candidate compound on the production of Axcex2. In a preferred embodiment, the method is used to identify inhibitors of the enzymatic production of Axcex2 from APP or a fragment thereof. The effect of the candidate compound on the production of Axcex2 may, for example, be monitored by measuring the amount of Axcex2 formed but other method of monitoring are also within the scope of the invention. In a preferred embodiment, the method is used to identify inhibitors that reduce the amount of Axcex2 formed by at least about 50%, preferably by at least about 75%, more preferably by at least about 85%, most preferably by at least about 90%.
Just as in the previous aspects, APP may, for example, be a native sequence human APP including the 695-amino acid isotype and the isotype containing the so called Swedish mutation. The APP fragment specifically includes, without limitation, xcex2-CTF . The method may be performed in the presence of an xcex1-secretase activity and/or a xcex3-secretase activity and/or a xcex2-secretase activity other than BACE2. The additional xcex2-secretase activity may, for example, be due to the presence of an enzyme having a pH optimum at about pH 6.5-7.0, and an estimated molecular weight of about 32-39 kDa as calculated from radiation inactivation analysis of HEK293 cell membrane extracts, or about 20-26 kDa as calculated from radiation inactivation analysis of human brain samples, with a candidate compound. Alternatively or in addition, the additional xcex2-secretase activity may be due to the presence of a xcex2-secretase enzyme having a pH optimum at about pH 4.5-5.0 and an estimated molecular weight of about 50-60 kDa as calculated from radiation inactivation analysis of HEK293 cell membrane extracts or human brain samples (BACE1).
In a still further aspect, the invention concerns a modulator of the enzymatic production of Axcex2-amyloid peptide (Axcex2) from Axcex2-amyloid precursor protein (APP) or a fragment thereof, identified by the foregoing method. The modulator preferably is an inhibitor, and may, for example, be a polypeptide, peptide, or small molecule.
In an additional aspect, the invention concerns a method for reducing the amount of Axcex2-amyloid deposits in the central nervous system (CNS) of a mammal comprising administering to the mammal an effective amount of BACE2 or an agonist thereof.
In another aspect, the invention concerns a method for the treatment of Alzheimer""s disease (AD), an AD-type pathology or cerebral amyloid angiopathy in a mammalian patient, comprising administering to the patient an effective amount of BACE2 or an agonist thereof.